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FIJI for Beginners Workshop
Dr Paul McMillan
Biological Optical Microscopy Platform
1
2
• Installation
• Image Import
• Image manipulation
– Duplicating
– Colour tools
– ROI tools
– Cropping
– Brightness & Contrast
– Look Up Tables (LUTs)
• Annotation
– Calibrating images
– Adding a scale bar
– Calibration bar
• 2D data
– Image panel
• 3D data
– Z-projection
– Orthogonal viewer
– Montage
– Projection movie
– Rotation Movie
• Timelapse data
– Timelapse movie
– Annotating movies
– Kymogram
Hands on Demonstrations
3
• Download from http://fiji.sc/#download
• Install on a drive you have admin rights over
• Plugins
• Update sites (FIJI/Help/Update
Installation
4
1. FIJI/File/open
2. Drag & drop files
3. FIJI/Plugins/bio-formats/bioformats importer
Image Import
5
Top of images gives us information about the image
The below example tells us:
c: 1/2 There are two channels in this images. We are looking at channel 1 at the moment.
z: 28/54 There are 54 z slices in this image. We are looking at slice number 28 at the moment.
Size: Our image is 40.96 microns in length/height
Pixels: The image is made up of 1024 x 1024 pixels
Bit-depth: The image is 32-bit
File size: The Image is 432 Mb
Image Information
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• Visualises a single parameter
• Open sixcolours.czi image
• Scroll through channels at the bottom left
Stacks
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• Visualises Multi-parameter data
• Open OMX.dv image
• Scroll through channels & Z positions
• We will modify this image for our visualisation
Hyperstacks
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FIJI Functions
Command Finder: If you are unsure where a function is located
Shortcuts
• Control + Shift L (PC)
• Command + Shift L (Mac)
• Help/Search (Mac)
Close All Images
Use File/Close All
Shortcut
• Control + Shift W (PC)
• Command + Shift W (Mac)
9
FIJI Functions
Image Type:
FIJI/Image/Type
e.g. Convert images to RGB format for use in office etc
Stacks:
FIJI/Image/Stacks
e.g. Stack to images = Turns stack into individual images
10
Resizing an image: Use the + or - buttons on your keyboard
Image Duplication: Always duplicate an image before making any modifications
• Select open image (or tab to select)
• FIJI/Image/duplicate
• Shortcut
• Command + Shift D (Mac)
• Control + Shift D (PC)
Image Resizing & Duplication
11
Colour Tools
FIJI/Image/Colour
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Colour Tools: Split Channels
FIJI/Image/Colour/Split Channels
Activity:
1) Open BPAE.tif
2) Split the channels
Optional:
1) Duplicate the images (keep the split images for next activity)
2) Change LUTs
3) Convert to RBG
4) Save as .tiff
13
Colour Tools: Merge Channels
FIJI/Image/Colour/Merge Channels
Activity:
1) Select BPAE folder
2) Open BPAE (blue).tif, BPAE (green).tif and BPAE (red).tif in FIJI
3) Select which colour you want to assign for each image
4) Click OK and save as .tif
14
Colour Tools: Arrange Channels
FIJI/Image/Colour/Arrange Channels
Activity:
1) Open 6 colours.czi file
2) Rearrange the order of the channels (as per your choice)
15
Colour Tools: Channels tool
FIJI/Image/Colour/Channels tool
Shortcut: Control + Shift Z
Activity:
1) Open Zeiss.lsm in FIJI
2) Open Channels tool dialog
3) Select colour option, select channel, click more and assign a LUT
4) Do this for all channels
5) Make Composite
6) Convert to RGB
7) Save as tif
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• Select open image
• Select rectangle tool
• Draw on image
Resizing ROIs (combinations can be used)
Shift – resizes symmetrically (same dimensions in x & y)
Control (PC), Command (Mac) - Free resize are centre of ROI
Alt – maintains aspect ratio on existing ROI
Define ROI size
FIJI/Edit/Selection/Specify
ROI tools
17
Making same ROI of multiple images
• Draw ROI on first image
• Select next image, FIJI/Edit/Selection/Restore Selection
• Shortcuts
• Command + Shift E (Mac)
• Control + Shift E (PC)
ROI Manager (more than one ROI on an single image)
• Select open image
• FIJI/Analyse/Tools/ROI manager
• Draw on ROI & click “Add”
• Click “Show All” view all selected ROI
ROI Tools
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Image Cropping
FIJI/Image/Crop
Shortcuts: Control + Shift X (PC) or Command + Shift X (Mac)
Activity:
1) Select open image
2) Duplicate the image
3) Draw an ROI (left click and hold)
4) FIJI/Image/Crop
• Can use Restore Selection (FIJI/edit/Selection/Restore Selection) to crop same area on multiple images
19
FIJI/Image/Lookup Tables or LUT shortcut
Activity:
1) Open an image from demo images (e.g. BPAE green.tif)
2) Click LUT
3) Select a LUT of your choice
4) Convert to RBG (Image/Type/RGB color)
5) Save image as tif
To visualize LUTs available
Image/Color/Display LUTs
Look Up Tables (LUTs)
20
Enables people looking at your image to interpret what the different coloursin the LUT mean
Activity:
1) Select the open file
2) Change LUT to fire
3) FIJI/Analyze/Tools/Calibration Bar
4) Select various options
5) Untick Overlay
6) Save as tif
Calibration Bar
21
Adjust for Visualisation purposes only (mention this in materials & methods)
Activity:
1) Select Zeiss.lsm image
2) Convert LUT to HiLo (Used to detect saturated (red) or clipped (Blue) pixels)
3) Move maximum and minimum only to (and not past) the data
4) FIJI/Image/Adjust/Brightness & Contrast
5) Shortcut
• Command + Shift C (Mac)
• Control + Shift C (PC)
Click Apply once you have changed the settings (original data is LOST!!)
Brightness & Contrast
22
Activity:
• Open “Leica triple merge (uncalibrated).tif” from demo images folder
• FIJI/Images/Properties
• Shortcuts
– Command + Shift P (Mac)
– Control + Shift P (PC)
• Enter the below numbers
• Unit of length = um
• Pixel width = 0.031678
We can assist you to measure your pixel sizes e.g.Calibration slide or Hemocytometer grid
Calibrating images
23
• Measure the pixel length over a known distance
• Images must be acquired EXACLTY the same way as your data (camera, magnification, pixel number, binning)
• Use the Line ROI to measure the distance in pixels
Calibrating images
24
• Select the open file
• FIJI/Analyse/Tools/Scale Bar
• Modify the options to suit
Adding a scale bar
25
Use all you now know to make a panel for a figure
Activity:
1) Open BPAE.tif
2) Duplicate image
3) Split the channels on one image
4) Assign the correct LUTs (if required)
5) Convert Images to stacks (Image/stacks/Images to Stacks, all images should be the same size)
6) Stack sorter (Image/Stacks/Stack Sorter) to re-order stacks
7) Make Montage (Image/Stack/Make Montage)
2D Data presentation
26
Figure J plugin (download via below update sites)
IBMP-CNRS
ImageScience
BioFormats
http://imagejdocu.tudor.lu/doku.php?id=plugin:utilities:figurej:start)
2D Data presentation
27
Activity:
1) Open “OMX (3D data).tif”
2) FIJI/Image/Stack/Z Project (or via shortcut above)
3) Select first & last slices to be used
4) Select projection type
• Maximum intensity = Brightest pixel (in Z) at each xy position
• Average intensity = Average intensity (in Z) at each xy position
3D data: Z projection
28
Activity:
1) Open “OMX (3D data).tif”
2) FIJI/Image/Stack/Orthogonal views
• Via STK shortcut
• Control + Shift H (PC)
• Command + Shift H (Mac)
3) Move cross hairs
4) Change focus
5) Save individual panels
3D data: Orthogonol viewer
29
Activity:
• Open “OMX (3D data)”
• FIJI/Image/Stack/Make Montage
• Update settings below
– Columns = 3
– Rows = 3
– Scale = 1
– First = 1
– Last = 54
– Increment = 5
– Border width = 3
3D data: Montage
30
Activity:
1) Open “OMX (3D data).tif”
2) FIJI/Image/Stack/Animation Options or Right click on play button
3) Select the option you require
4) FIJI/Save As/avi
5) Set fps and select uncompressed if you want to convert to a different file type
3D data: Z translation
31
Activity:
• Open “OMX (3D data).tif”
• FIJI/Image/Stack/3D Project
• Try varying some of these options
– Projection method
– Axis of rotation
– Total rotation
– Rotation increments
– Interpolate
• Save as .avi as before
3D data: Rotation Movie
32
Timelapse data: Movie
Activity:
• Open “ER live.tif”
• Apply green LUT
• FIJI/Image/Stack/Animation Options or Right click on play button
• Select the options you require
• FIJI/Save As/avi
• Set fps and select uncompressed if you want to convert to a different file type
33
Images were taken every 4 seconds
Activity:
• Open “ER live.tif”
• FIJI/Images/Stacks/Label
• Try changing the below parameters
– Format
– Interval
– XY location
– Font size
• Text colour can be changed at FIJI/Image/Colours/Colour Picker
• Save as avi as before
Annotating movies
34
Activity:
1) Open tracking.tif from Kymogram
2) Image/Colour/Split channels
3) Draw line form ROI tools (Hold shift to keep it straight)
4) Select channel two and restore selection (Ctrl + Shift E)
5) Select channel 1, Analyze/Multi Kymporaph/Multi Kymograph
6) Set line width to 29 (must be an odd number)
7) Image/Rename, rename channel 1
8) Repeat Kymograph (as above) for Channel 2
9) Merge Channels (Channel 1 = green, Channel 2 = Red)
Kymogram
35
• Installation
• Image Import
• Image manipulation
– Duplicating
– Colour tools
– ROI tools
– Cropping
– Brightness & Contrast
– Look Up Tables (LUTs)
• Annotation
– Calibrating images
– Adding a scale bar
– Calibration bar
• 2D data
– Image panel
• 3D data
– Z-projection
– Orthogonal viewer
– Montage
– Projection movie
– Rotation Movie
• Timelapse data
– Timelapse movie
– Annotating movies
– Kymogram
Hands on Demonstrations